Search

Rationale: Extracellular vesicles (EVs) contain bioactive cargo including microRNAs (miRNAs) and proteins that are released by cells as a form of cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels and thereby interface with cells in the circulation as well as cells residing in the vascular wall. It is unknown whether ECs have the capacity to release EVs capable of governing recipient cells within two separate compartments, and how this is affected by endothelial activation commonly seen in atheroprone regions.Objective: Given their boundary location, we propose that ECs utilize bidirectional release of distinct EV cargo in quiescent and activated states to communicate with cells within the circulation and blood vessel wall.Methods and Results: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1β activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis. RNA sequencing of EV-treated monocytes and smooth muscle cells (SMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic. Apical and basolateral EV release was assessed using ECs on transwells. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis.In silicoanalysis determined that compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and SMCs, respectively.Conclusions: The demonstration that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance our ability to design endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.Non-standard Abbreviations and Acronymscryo-EMcryogenic electron microscopyECendothelial cellEVextracellular vesicleGOgene ontologyHAEChuman aortic endothelial cellSMChuman aortic vascular smooth muscle cellIL-1βinterleukin 1 betaKEGGKyoto encyclopedia of genes and genomesLC-MSlabel-free liquid-chromatography mass spectrometryMVBmultivesicular bodymiRNAmicroRNARNAseqRNA sequencingTEMtransmission electron microscopyTIRFtotal interal reflection fluorescence microscopymiRNAmicroRNAGraphical abstract: Polarized endothelial extracellular vesicle communication with luminal and abluminal vascular cellsEndothelial cell small extracellular vesicle (EC-EV) release from apical (luminal) and basolateral (abluminal) surfaces in quiescence and after endothelial activation. Quiescent EC-EVs are depicted in blue (bright blue=apical, light blue=basolateral), while activated EC-EVs are depicted in red (bright red=apical, light red=basolateral). Luminal monocyte is represented in purple with upregulation of pro-inflammatory transcripts (bright purple) after uptake of activated EC-EVs from the apical surface, compared to uptake of quiescent apical EC-EVs (light purple). Basolateral EC-EVs are taken up by an abluminal resident smooth muscle cell depicted in yellow. Smooth muscle cell uptake of activated basolateral EC-EVs with upregulation of pro-inflammatory/pro-atherogenic transcripts (bright yellow), as compared to uptake of quiescent EC-EVs (light yellow).

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is an essential and evolutionarily conserved transcription factor complex primarily involved in innate immunity and inflammation. Transposable elements (TEs) can be co-opted to innovate immune transcriptional regulatory networks; however, the extent to which TEs have contributed to the modulation of NF-κB response in different mammalian lineages is not well established. Here we performed a multi-species analysis of TEs bound by the NF-κB subunit RELA (p65) in response to the pro-inflammatory cytokine TNFα (Tumor Necrosis Factor alpha). Using endothelial cell RELA ChIP-seq data from human, mouse and cow, we found that 55 TE subfamilies were enriched within NF-κB bound regions. These RELA-bound transposons possess multiple active epigenetic features and reside near TNFα-induced genes. A prominent example of lineage-specific contribution of transposons comes from the bovine SINE subfamilies Bov-tA1/2/3 which collectively contributed over 14,000 NF-κB bound regions in cow. By comparing NF-κB binding data across species, we found several examples of NF-κB motif-bearing TEs that appeared to colonize the genome prior to the divergence of the selected mammals, including a DNA transposon MER81, whose ancestral sequence contains two intact RELA motifs. We demonstrate that one NF-κB bound MER81 element can control the TNFα-induced expression ofINFGR2(Interferon Gamma Receptor 2) in human. Lastly, the presence of RELA motifs within MER81 elements appeared to stabilize during human evolution, indicative of purifying selection acting on a subset of these NF-κB bound ancient DNA transposons. Taken together, our results implicate multiple transposons in establishing NF-κB mediated regulatory networks during mammalian evolution.

Liver failure causes blood-brain-barrier (BBB) breakdown leading to central nervous system damage, however the mechanisms whereby the liver influences BBB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BBB integrity. We developed light-sheet imaging for three-dimensional study of BBB function. We show that liver- or muscle-specific knockout of Hfe2 induces BBB breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits. In healthy animals, soluble Hfe2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells and the ensuing BBB disruption. Hfe2 administration in an animal model of multiple sclerosis prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BBB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BBB dysfunction such as multiple sclerosis.

Rationale: We previously identified somatic activating mutations in the KRAS ( Kirsten rat sarcoma viral oncogene homologue ) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. Objective: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. Methods and Results: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. Conclusions: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Objective: Coronavirus disease 2019 (COVID-19) is a global pandemic involving >5 500 000 cases worldwide as of May 26, 2020. The culprit is the severe acute respiratory syndrome coronavirus-2, which invades cells by binding to ACE2 (angiotensin-converting enzyme 2). While the majority of patients mount an appropriate antiviral response and recover at home, others progress to respiratory distress requiring hospital admission for supplemental oxygen. In severe cases, deterioration to acute respiratory distress syndrome necessitating mechanical ventilation, development of severe thrombotic events, or cardiac injury and dysfunction occurs. In this review, we highlight what is known to date about COVID-19 and cardiovascular risk, focusing in on the putative role of the endothelium in disease susceptibility and pathogenesis. Approach and Results: Cytokine-driven vascular leak in the lung alveolar-endothelial interface facilitates acute lung injury in the setting of viral infection. Given that the virus affects multiple organs, including the heart, it likely gains access into systemic circulation by infecting or passing from the respiratory epithelium to the endothelium for viral dissemination. Indeed, cardiovascular complications of COVID-19 are highly prevalent and include acute cardiac injury, myocarditis, and a hypercoagulable state, all of which may be influenced by altered endothelial function. Notably, the disease course is worse in individuals with preexisting comorbidities that involve endothelial dysfunction and may be linked to elevated ACE2 expression, such as diabetes mellitus, hypertension, and cardiovascular disease. Conclusions: Rapidly emerging data on COVID-19, together with results from studies on severe acute respiratory syndrome coronavirus-1, are providing insight into how endothelial dysfunction may contribute to the pandemic that is paralyzing the globe. This may, in turn, inform the design of biomarkers predictive of disease course, as well as therapeutics targeting pathogenic endothelial responses.

We have adapted a zebrafish (Danio rerio) tumor xenograft model for use in the study of oncolytic virotherapy. Following implantation of mammalian cancer cells into the perivitelline space of developing zebrafish embryos, both local and intravenous oncolytic virus treatments produce a tumor-specific infection with measurable antitumor effects. Tumor cells are injected at 48 h post fertilization, with oncolytic virus treatment then being administered 24 h later to allow for an initial period of tumor development and angiogenesis. Confocal fluorescent imaging is used to quantify dynamics within the tumor environment. The natural translucency of zebrafish at the embryo stage, coupled with the availability of strains with fluorescent immune and endothelial cell reporter lines, gives the model broad potential to allow for real time, in vivo investigation of important events within tumors throughout the course of virotherapy. Zebrafish xenografts offer a system with biologic fidelity to processes in human cancer development that influence oncolytic virus efficacy, and to our knowledge this is the first demonstration of the model’s use in the context of virotherapy. Compared with other models, our protocol offers a powerful, inexpensive approach to evaluating novel oncolytic viruses and oncolytic virus-based combination therapies, with potential application to investigating the impacts of virotherapy on immune response, tumor vasculature, and metastatic disease.

Coronary artery disease is one of the major causes of morbidity and mortality worldwide. Coronary stents, tube-shaped medical implants that are placed in narrowed coronary arteries, have been used successfully in the management of this condition. However, re-narrowing (i.e. restenosis) of the artery can occur which is instigated by an immune response towards the implanted ‘foreign’ material. A new approach to prevent restenosis and reduce the stent-induced immune response has been proposed previously, which involves re-endothelialization of the implanted stent. In the present study a proof-of-concept experiment involving surface-modified medical grade steel was employed in order to examine the best surface chemistry for in vitro cell capture. Steel coupons were coated with a silanized adlayer, followed by attachment of whole antibodies, followed by culturing of human umbilical vein endothelial cells (HUVECs) or human aortic endothelial cells (HAECs or HAoECs). With regard to the adlayer-antibody configuration, HUVECs adhered and grew with normal morphology, and a significantly increased number of HUVECs proliferated on the coupons compared to the ‘bare’ surface. Similar effects were observed with HAECS grown on adlayer-antibody modified substrates, with a significantly higher number of cells proliferating. These results demonstrate a successful strategy for re-endothelialization of the steel surface that may prevent immune response with respect to the behaviour of steel-based stents in vivo.

Background: Stroke is a devastating consequence of plaque rupture from the carotid arteries. Current management of carotid plaques involves waiting for symptoms (e.g., stroke or mini-stroke), as intervention itself has risk of stroke and not all plaques are vulnerable to rupture. There is a need to better risk-stratify plaque that causes stroke. Carotid ultrasound (US) is a non-invasive and inexpensive visualization of plaques but is limited by human interpretation. We hypothesize that convolutional neural networks (CNNs) will identify unique features of carotid plaques for automated risk stratification. Methods: Our workflow is illustrated in Fig. a. A total of 141 B-mode US images of carotid arteries were included; 64 high-risk with symptomatic carotid plaques and 75 low-risk with no significant plaque. Data was cropped and divided into training (70%) and holdout test (30%) subsets. During model training, an ensemble of ResNet-18 CNNs learned classification of low-risk and high-risk cases using five-fold stratified cross validation and was used to predict on the holdout test set. The model was evaluated using ROC-AUC and sensitivity. Saliency maps were used for model interpretability to highlight relevant pixels for model decisions. Results: The cross-validation AUC was 0.995 ± 0.010. The testing AUC was 0.909 and class-wise sensitivities were 0.88 (low-risk) and 0.79 (high-risk). The density plot (Fig. b) shows that the classifier correctly identifies both classes with confidence. Model interpretability using saliency maps (Fig. c) shows pixels corresponding to carotid artery vessel edges and in high-risk cases, carotid plaques. Conclusions: Using this proof-of-concept model, carotid US long axis images are sufficient to identify high-risk plaques in symptomatic patients - we now need to determine whether we can identify high-risk plaques before symptoms to prevent devastating stroke caused by carotid disease.

Aim: Abdominal aortic aneurysm (AAA) contributes to significant post-rupture mortality in aging populations. AAA management with watchful waiting and surgical repair is based on our limited understanding of disease processes, and current research foci including extracellular vesicles (EVs; nano-sized packages of proteins, RNAs, and lipids that facilitate intercellular communication) may aid in developing novel AAA therapies. To characterize this regulatory cargo, we isolated EVs from human AAA tissue or control aortic punch biopsies and profiled EV content with microRNA (miRNA) sequencing to identify dysregulated pathways. Methods: The study was approved by the University Health Network Research Ethics Board. EVs were isolated from human AAA or aortic punch tissue and enriched using size exclusion chromatography (SEC; qEVoriginal columns 70 nm, Izon Science Ltd.) (n=3). EV size and concentration were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical Ltd.). EV-miRNA sequencing was performed with Illumina NextSeq (HTG Molecular Diagnostics Inc.) and analyzed using Partek Genomics Suite (v.10) and MIENTURNET (19-11-25). Results: Patients were selected for infrarenal AAA requiring surgical repair (AAA) or coronary artery disease requiring bypass graft surgery (control). EV size and concentration were confirmed with NTA. Principal components and gene set analyses revealed distinct clustering of tissue types with 901 and 687 miRNAs enriched in AAA and control samples, respectively. Pathway prediction using established AAA miRNAs (e.g., miR-122, miR-146a, and miR-503) identified significant interactions with proaneurysmal signaling pathways (e.g., PI3K-AKT, JAK-STAT, and HIF-1) as well as cell senescence and adhesion processes (FDR < 0.05). Conclusion: EV-derived miRNAs from patients with AAA prominently associate with cell signaling, senescence, and adhesion pathways in aneurysm pathogenesis. To our knowledge, this is the first study to profile the EV-miRNA landscape in human AAA tissue. Further investigation will explore EV-miRNAs as mediators of communication between distinct vascular cell populations that contribute to AAA development.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Background Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction. Methods The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs). Results We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid β-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid β-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium. Conclusions MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart.

Aims: The transcription factor ERG has emerged as an important regulator of vascular function through its ability to repress inflammation in endothelial cells (ECs) and ERG dysregulation is associated with chronic inflammation in atherosclerosis and aortic aneurysms. To characterize the cellular mechanisms that underlie this regulation, we deleted ERG in human aortic ECs and assessed genome-wide chromatin accessibility and gene expression to identify dysregulated pathways. Furthermore, we have begun to investigate ERG expression in mouse atherosclerotic plaque. Methods: CRISPR/Cas9 was used to delete ERG exon 6 in immortalized human aortic ECs. Wildtype or Δ ERG ECs were profiled with ATAC-seq and RNA-seq (n=2-4). GREAT and clusterProfiler were used to identify enriched pathways (log 2 FC>|1|; FDRLdlr −/− mice were fed a 12-week high-cholesterol diet (1.25%) before sacrifice. Ascending aortic sections were stained with anti-CD68 and anti-ERG (n=2). Images were obtained using a Leica SP8 confocal microscope. Results: Pathway analysis of >21,000 differentially accessible chromatin regions identified 67 enriched processes in Δ ERG ECs. Of these, the top 20% included disrupted wound healing, abnormal aorta morphology, and aneurysm, which were driven by association with proatherogenic and proaneurysmal genes including PDGFRB and TGFBR1 . Gene expression analysis revealed that loss of ERG contributes to endothelial to mesenchymal transition (increased KLF4 and SNAI2 ), inflammation (increased IL18 ), and dysregulated extracellular matrix and cell adhesion (increased FBLN2 and VCAN ). In the mouse aorta, preliminary data suggest that ERG expression may be elevated in EC nuclei overlying plaque compared to non-atherosclerotic regions. Conclusions: The loss of ERG has a broad impact on chromatin accessibility and gene expression in aortic ECs, with enrichment of vascular dysfunction pathways. Further investigation will establish how altered ERG expression contributes to in vivo models of atherosclerosis and aortic aneurysms.

Oral tyrosine kinase inhibitors have revolutionized the treatment of chronic myelogenous leukemia, with many patients achieving major clinical and molecular responses without complications. While typically well-tolerated, clinical experience with tyrosine kinase inhibitors (particularly those of the second and third generations) has highlighted unanticipated associations with serious non-cancer adverse effects on various organs, particularly the cardiovascular system. Herein, we review the current literature surrounding the major cardiovascular toxicities of BCR-ABL1 tyrosine kinase inhibitors in chronic myelogenous leukemia, discuss potential mechanisms underpinning their development, and suggest future research directions to uncover novel ways to reduce cardiovascular events in patients treated with tyrosine kinase inhibitors. As a whole, while cardiovascular toxicities are well-documented, the mechanistic basis of these clinical observations remains poorly defined. In turn, to provide safe and effective treatment to all patients, it is necessary to close the knowledge gap regarding mechanisms that drive toxicity and elucidate the complex interactions that predispose specific individuals to these toxicities.

Significant improvements in cancer survival have brought to light unintended long-term adverse cardiovascular effects associated with cancer treatment. Although capable of manifesting a broad range of cardiovascular complications, cancer therapy-related cardiac dysfunction (CTRCD) remains particularly common among the mainstay anthracycline-based and human epidermal growth factor receptor-targeted therapies. Unfortunately, the early asymptomatic stages of CTRCD are difficult to detect by cardiac imaging alone, and the initiating mechanisms remain incompletely understood. More recently, circulating inflammatory markers, cardiac biomarkers, microRNAs, and extracellular vesicles (EVs) have been considered as early markers of cardiovascular injury. Concomitantly, the role of the endothelium in regulating cardiac function in the context of CTRCD is starting to be understood. In this review, we highlight the impact of breast cancer therapies on the cardiovascular system with a focus on the endothelium, and examine the status of circulating biomarkers, including inflammatory markers, cardiac biomarkers, microRNAs, and endothelial cell-derived EVs. Investigation of these emerging biomarkers may uncover mechanisms of injury, detect early stages of cardiovascular damage, and elucidate novel therapeutic approaches.

We have adapted a zebrafish (Danio rerio) tumor xenograft model for use in the study of oncolytic virotherapy. Following implantation of mammalian cancer cells into the perivitelline space of developing zebrafish embryos, both local and intravenous oncolytic virus treatments produce a tumor-specific infection with measurable antitumor effects. Tumor cells are injected at 48 h post fertilization, with oncolytic virus treatment then being administered 24 h later to allow for an initial period of tumor development and angiogenesis. Confocal fluorescent imaging is used to quantify dynamics within the tumor environment. The natural translucency of zebrafish at the embryo stage, coupled with the availability of strains with fluorescent immune and endothelial cell reporter lines, gives the model broad potential to allow for real time, in vivo investigation of important events within tumors throughout the course of virotherapy. Zebrafish xenografts offer a system with biologic fidelity to processes in human cancer development that influence oncolytic virus efficacy, and to our knowledge this is the first demonstration of the model's use in the context of virotherapy. Compared with other models, our protocol offers a powerful, inexpensive approach to evaluating novel oncolytic viruses and oncolytic virus-based combination therapies, with potential application to investigating the impacts of virotherapy on immune response, tumor vasculature, and metastatic disease.

STRUCTURED ABSTRACTBackgroundEndothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe COVID-19, indicating that the vasculature is affected during the acute stages of SARS-CoV-2 infection. It remains unknown whether circulating vascular markers are sufficient to predict clinical outcomes, are unique to COVID-19, and if vascular permeability can be therapeutically targeted.MethodsEvaluating the prevalence of circulating inflammatory, cardiac and EC activation markers, and the development of a microRNA atlas in 241 patients with suspected SARS-CoV-2 infection, allowed their prognostic value to be assessed by a Random Forest model machine learning approach. Subsequent ex vivo experiments assessed EC permeability responses to patient plasma and were used to uncover modulated gene regulatory networks from which rational therapeutic design was inferred.FindingsMultiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients, while dysregulation of specific microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating the datasets using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction which was ameliorated using angiopoietin-1 mimetic or recombinant Slit2-N.InterpretationIntegration of multi-omics data identified microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and revealed that vascular stabilizing therapies should be explored as a treatment for endothelial dysfunction in COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis.RESEARCH IN CONTEXTEvidence before this studyWhile diagnostic testing has allowed for the rapid identification of COVID-19 cases, the lack of post-diagnosis risk assessment metrics, especially among the highest-risk subgroups, thereby undermined the cascade and allocation of care. To date, the integration of clinical data with broad omics technologies has opened up new avenues for efficiently delineating complex patient phenotypes and their associations with clinical outcomes, with circulating profiles of plasma microRNAs (miRNA), in particular, having been shown to be tightly associated with disease, and capable of providing not only detailed prognostic information but also mechanistic insight.Added value of this studyMarkers of endothelial dysfunction at presentation, while indicative of poor outcomes in COVID-19-positive patients, likely reflect systemic vascular dysfunction in critically ill patients and are not specific to SARS-CoV-2 infection. More so, the generation of a plasma microRNA atlas uncovers COVID-19-specific prognostic markers and multiple disease-specific pathways of interest, including endothelial barrier dysfunction. Furthermore, synthesis of electronic health record data with clinically relevant multi-omic datasets using a machine learning approach provides substantially better metrics by which mortality can be estimated in patients with severe COVID-19. Finally, targeted stabilization of the endothelial barrier with Q-Peptide and Slit2-N are novel therapeutic avenues that should be explored in COVID-19 patients.Implications of all the available evidenceTogether, our work provides biological insight into the role of the endothelium in SARS-CoV-2 infection, the importance of miRNA as disease- and pathway-specific biomarkers, and the exciting possibility that endothelial barrier stabilizing treatments might hold promise in COVID-19.

Background Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. Methods We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations,...

Objective— During inflammation, macrophages secrete vesicles carrying RNA, protein, and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis; however, the role of macrophage-derived EVs in atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNAs) in EVs to mediate cell–cell communication and promote proinflammatory and proatherogenic phenotypes in recipient cells. Approach and Results— We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized low-density lipoprotein) and characterized the EV miRNA expression profile. We confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365, and miR-503 in atherogenic EVs compared with controls and demonstrate that these EVs are taken up and transfer exogenous miRNA to naive recipient macrophages. Bioinformatic pathway analysis suggests that atherogenic EV miRNAs are predicted to target genes involved in cell migration and adhesion pathways, and indeed delivery of EVs to naive macrophages reduced macrophage migration both in vitro and in vivo. Inhibition of miR-146a, the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1) and HuR (human antigen R or ELAV-like RNA-binding protein 1) in recipient cells, and knockdown of IGF2BP1 and HuR using short interfering RNA greatly reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. Conclusions— EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)–derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr −/− (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr −/− ;miR-146a −/− mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr −/− mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1( Sort1 ), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) represent a cellular hub coordinating various cellular processes that are critical in health and disease. Mechanical stress triggers changes in vascular smooth muscle cells (VSMCs) that in turn contribute to pathophysiological changes within the vasculature. We sought to evaluate the role that lncRNAs play in mechanical stretch-induced alterations of human aortic smooth muscle cells (HASMCs). RNA (lncRNA and mRNA) samples isolated from HASMCs that had been subjected to 10 or 20% elongation (1 Hz) for 24 h were profiled with the Arraystar Human LncRNA Microarray V3.0. LncRNA expression was quantified in parallel via qRT-PCR. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (DE, P

Stroke is the leading cause of serious disability in the world and a large number of ischemic strokes are due to thromboembolism from unstable carotid artery atherosclerotic plaque. As it is difficult to predict plaque rupture and surgical treatment of asymptomatic disease carries a risk of stroke, carotid disease continues to present major challenges with regard to clinical decision-making and revascularization. There is therefore an imminent need to better understand the molecular mechanisms governing plaque instability and rupture, as this would allow for the development of biomarkers to identify at-risk asymptomatic carotid plaque prior to disease progression and stroke. Further, it would aid in creation of therapeutics to stabilize carotid plaque. MicroRNAs (miRNAs) have been implicated as key protagonists in various stages of atherosclerotic plaque initiation, development and rupture. Notably, they appear to play a crucial role in carotid artery thromboembolism. As the molecular pathways governing the role of miRNAs are being uncovered, we are learning that their involvement is complex, tissue- and stage-specific, and highly selective. Notably, miRNAs can be packaged and secreted in extracellular vesicles (EVs), where they participate in cell–cell communication. The measurement of EV-encapsulated miRNAs in the circulation may inform disease mechanisms occurring in the plaque itself, and therefore may serve as sentinels of unstable plaque as well as therapeutic targets.

While interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.

Significance Controlling vascular inflammation is critical for limiting the progression of chronic vascular diseases such as atherosclerosis. Although poorly studied in the context of human vascular inflammation, long noncoding RNAs (lncRNAs) have the potential to regulate their neighboring genes. However, what constitutes a neighboring lncRNA is currently not well defined. In this study, we took an innovative approach to define IL-1β−regulated neighboring mRNA−lncRNA pairs based on colocalization within the same chromatin neighborhood and divergent transcriptional orientation. This approach led to the discovery of lncRNA-CCL2, which positively regulates its neighboring gene, CCL2, an important player in atherogenesis. Furthermore, lncRNA-CCL2 is relevant to human disease, as it is elevated in human atherosclerotic plaques, and, given its regulatory role, it may contribute to atherogenesis., Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)−regulated messenger RNA (mRNA)−lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2, which is transcribed divergently to the gene, CCL2, encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA−lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2, which may contribute to atherogenesis in humans.

While interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63+ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body., Senescent cells accumulate in the body with aging and alter circulating extracellular vesicle microRNA content. This can be replicated in young mice by inducing senescence using sub‐lethal irradiation. Conversely, removal of senescent cells in aged mice by senolytic treatment restores circulating extracellular vesicle microRNA expression to that of a younger phenotype.

Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)−regulated messenger RNA (mRNA)−lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2 , which is transcribed divergently to the gene, CCL2 , encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA−lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2 , which may contribute to atherogenesis in humans.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor plays a prominent role in inflammation and contributes to the development of atherosclerosis. Genome-wide DNA binding assays of the human NF-κB subunit RELA (p65) have revealed tens of thousands of NF-κB binding sites and hundreds of target genes. However, the function of individual RELA binding sites and the extent to which NF-κB occupancy and function is conserved across mammals are not well understood. To better understand the function of NF-κB we characterized the genome-wide binding of RELA in primary vascular endothelial cells (ECs) isolated from the aortas of human, mouse and cow. ECs were stimulated acutely with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA) and we profiled RELA occupancy, open chromatin, select histone modifications, and RNA expression. We found ~5000 RELA binding events conserved across all three species and these highly conserved human binding events were enriched for genes controlling vascular development, apoptosis, and pro-inflammatory responses. Approximately 2000 of these highly conserved RELA binding events were also shared across multiple human cell types, revealing a conserved core of robustly bound NF-κB sites. These NF-κB binding sites were also prominent components of ~40 inflammation-induced super-enhancers (SE) common to several tissues. To gain insight into the function of individual conserved NF-κB binding sites we focused on the inflammation-induced SE proximal to the monocyte recruiting chemokine CCL2 , which we detected as a SE in all three species and across multiple cell types. We tested the functional significance of six conserved RELA binding sites comprising this SE using CRISPR/Cas9 genome editing. We found that only deletion of the most proximal upstream RELA binding site could abolish the induction of CCL2 upon TNFA treatment. This site also contains a disease associated variant that can modulate CCL2 induction. Overall, our comparative genomics assessment of NF-κB binding gives new insight into NF-κB biology and the function of conserved transcription factor binding events within mammalian super-enhancers.

Vascular inflammation is a critical driver of chronic diseases such as atherosclerosis. A network of NF-κB-dependent leukocyte adhesion molecules and chemokines are induced in endothelial cells (ECs) in response to inflammatory mediators. This includes chemokine (C-C motif) ligand 2 ( CCL2 ), which contributes to atherosclerosis by recruiting monocytes to the endothelium. Recently, long noncoding RNAs (lncRNAs) have been implicated in regulating gene expression through epigenetic mechanisms, but lncRNAs remain poorly studied in the context of vascular inflammation and NF-kB pathway regulation. LncRNAs are frequently retained in the nucleus where they interact with chromatin remodelling complexes to modulate the expression of neighboring protein-coding genes. Hence, identifying NF-kB-regulated neighboring mRNA-lncRNA pairs in vascular endothelial cells may uncover functional lncRNAs that play a role in fine-tuning the expression of their neighboring inflammatory genes. The Arraystar human lncRNA microarray V3 was employed to identify differentially expressed lncRNAs and mRNAs in ECs stimulated with the pro-inflammatory cytokine, IL-1β. Neighboring IL-1β-regulated mRNA-lncRNA pairs demonstrated a larger magnitude of mRNA induction than mRNAs lacking a neighboring lncRNA. This phenomenon was associated with shared regulatory elements and localization within the same topologically associated domain. Follow-up analysis was performed on the nuclear-enriched, lncRNA-CCL2 , which is transcribed through a super-enhancer near CCL2 . Both lncRNA-CCL2 and CCL2 responded to the same inflammatory stimuli. Similar to CCL2 , lncRNA-CCL2 transcript was elevated in unstable human atherosclerotic plaques. Knockdown of lncRNA-CCL2 decreased CCL2 mRNA levels in multiple EC cell lines, but had no effect on other inflammatory genes or distal CCL genes. Hence, our approach has uncovered neighboring IL-1β-regulated mRNA-lncRNA pairs and identified a novel functional lncRNA, lncRNA-CCL2 .

Cells contributing to atherosclerotic disease are highly plastic and can shift their phenotype in a changing microenvironment. A study in Nature Metabolism now reveals that transforming growth factor-β (TGF-β) can transform endothelial cells into pro-inflammatory cells and that inhibition of TGF-β-receptor signalling in the endothelium can reverse atherosclerosis in mice.

Apolipoprotein E (apoE) has long been recognized as an important regulator of plasma lipoprotein metabolism and cholesterol homeostasis. However, despite decades of research into the biology of apoE, new aspects of its function continue to emerge. In this issue, Li et al1 demonstrate that apoE regulates the expression of an anti-inflammatory microRNA, miR-146a, in monocytes/macrophages. The authors reveal that miR-146a represses nuclear factor-κB (NF-κB) signaling in these cells and that intravascular delivery of miR-146a mimetics can inhibit atherogenesis in mouse models. These findings establish that enhancing miR-146a expression can antagonize atherogenesis and provide an impetus to pursue microRNA-based therapies for vascular inflammatory diseases. Article, see p e1 Initial work that characterized apoE function in the cardiovascular system demonstrated that apoE mediates the uptake of chylomicron remnants and triglyceride-rich very low and intermediate density lipoproteins in the liver. In arteries, together with apoA-I, apoE facilitates cellular cholesterol efflux from macrophage foam cells.2 Genetically engineered deficiency of Apoe in mice results in elevated plasma very low and intermediate density lipoproteins and spontaneous atherogenesis.3,4 Over the past 2 decades, this model has served as the mainstay of atherosclerosis research. Global Apoe deficiency can be rescued by transplantation of wild-type bone marrow, which normalizes plasma cholesterol levels and dramatically reduces atherosclerosis.5,6 Attenuated atherosclerosis is also observed in Apoe -null mice bred with transgenic mice expressing human apoE specifically in macrophages. These mice have plasma cholesterol levels comparable with control littermates.7 Interestingly, a recent study revealed that global hypomorphic expression of apoE has no effect on plasma cholesterol, yet atherosclerosis is suppressed and the expression of monocyte and endothelial cell inflammatory genes are reduced relative to complete Apoe deficiency in the low-density lipoprotein receptor–deficient ( Ldlr −/−) background.8 These studies highlight the importance of apoE in …

Autophagy in endothelial cells is an emerging anti-inflammatory and atheroprotective mechanism. In this issue of Circulation Research , Pankratz et al1 describe the function of an endothelial-enriched microRNA, miR-100, that promotes autophagy through direct suppression of the autophagy inhibitors, mTOR (mammalian target of rapamycin) and Raptor. Furthermore, they demonstrate that miR-100 suppresses proinflammatory NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and endothelial cell activation in an autophagy-dependent manner. Global inhibition of miR-100 in an in vivo model of atherosclerosis worsens disease while the opposite is the case when miR-100 mimetics are delivered intravenously. This study implies that therapeutics that can augment miR-100 levels in the endothelium will enhance autophagy and may be used to restrain vascular inflammation and atherogenesis. However, improvements in the specificity of microRNA delivery in vivo will be required to further test this concept because the authors also identify additional effects on cholesterol metabolism (albeit beneficial) in the livers of miR-100 mimetic-injected mice that seem to be autophagy dependent. Article, see p 417 The activation of the endothelium and subsequent recruitment of circulating inflammatory cells into the vessel wall is a central driver of atherosclerotic disease. This chronic vascular inflammatory response is primarily orchestrated by the transcriptional induction of a network of adhesion molecules, cytokines, and chemokines through the activity of the transcription factor, NF-κB, which is activated downstream of cholesterol accumulation in the vessel wall, and in response to disturbed blood flow patterns. Identifying the mechanisms that control the activation of NF-κB may reveal new therapeutic targets to block disease progression. However, it is important to note that targeting this pathway may only be effective in particular cell types or at specific stages of disease. For example, although endothelial NF-κB is required for atherosclerotic progression,2 suppression of macrophage NF-κB paradoxically accelerates atherosclerosis. …

The CCCTC-binding factor (CTCF) is a versatile transcriptional regulator required for embryogenesis, but its function in vascular development or in diseases with a vascular component is poorly understood. Here, we found that endothelial Ctcf is essential for mouse vascular development and limits accumulation of reactive oxygen species (ROS). Conditional knockout of Ctcf in endothelial progenitors and their descendants affected embryonic growth, and caused lethality at embryonic day 10.5 because of defective yolk sac and placental vascular development. Analysis of global gene expression revealed Frataxin (Fxn), the gene mutated in Friedreich's ataxia (FRDA), as the most strongly down-regulated gene in Ctcf-deficient placental endothelial cells. Moreover, in vitro reporter assays showed that Ctcf activates the Fxn promoter in endothelial cells. ROS are known to accumulate in the endothelium of FRDA patients. Importantly, Ctcf deficiency induced ROS-mediated DNA damage in endothelial cells in vitro, and in placental endothelium in vivo. Taken together, our findings indicate that Ctcf promotes vascular development and limits oxidative stress in endothelial cells. These results reveal a function for Ctcf in vascular development, and suggest a potential mechanism for endothelial dysfunction in FRDA.

The formation of a hierarchical vascular network, composed of arteries, veins, and capillaries, is essential for embryogenesis and is required for the production of new functional vasculature in the adult. Elucidating the molecular mechanisms that orchestrate the differentiation of vascular endothelial cells into arterial and venous cell fates is requisite for regenerative medicine, as the directed formation of perfused vessels is desirable in a myriad of pathological settings, such as in diabetes and following myocardial infarction. Additionally, this knowledge will enhance our understanding and treatment of vascular anomalies, such as arteriovenous malformations (AVMs). From studies in vertebrate model organisms, such as mouse, zebrafish, and chick, a number of key signaling pathways have been elucidated that are required for the establishment and maintenance of arterial and venous fates. These include the Hedgehog, Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β), Wnt, and Notch signaling pathways. In addition, a variety of transcription factor families acting downstream of, or in concert with, these signaling networks play vital roles in arteriovenous (AV) specification. These include Notch and Notch-regulated transcription factors (e.g., HEY and HES), SOX factors, Forkhead factors, β-Catenin, ETS factors, and COUP-TFII. It is becoming apparent that AV specification is a highly coordinated process that involves the intersection and carefully orchestrated activity of multiple signaling cascades and transcriptional networks. This review will summarize the molecular mechanisms that are involved in the acquisition and maintenance of AV fate, and will highlight some of the limitations in our current knowledge of the molecular machinery that directs AV morphogenesis.

During inflammation, macrophages secrete vesicles carrying RNA, protein and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis, however, the role of macrophage-derived EVs in promoting atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNA) in EVs to mediate cell-cell communication and promote pro-inflammatory and pro-atherogenic phenotypes in recipient cells. Results: We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized LDL) and characterized the EV-derived miRNA expression profile. Using microarrays and Q-PCR, we confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365 and miR-503 in atherogenic EVs compared to controls. Live-cell imaging demonstrated that macrophage-derived EVs are taken up and transfer exogenous miRNA ( C. elegans) to naive recipient macrophages. Bioinformatic analysis suggests that atherogenic EV-derived miRNAs are predicted to target genes involved in cell migration and adhesion pathways. Indeed, treatment of naïve macrophages with EVs from atherogenic but not control macrophages inhibited the migration of naïve cells towards a chemokine stimulus (80% decrease in migration, p≤0.01). In vivo , delivery of EVs also abolished LPS-induced emigration of mouse peritoneal macrophages . Moreover, inhibition of miR-146a (using anti-miR oligonucleotides or miR-146a -/- macrophages), the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 and HuR in recipient cells, and knockdown of IGF2BP1 and HuR using siRNA reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. Notably, expression of miR-146a was elevated in mouse and human atherosclerotic lesions compared to controls. Thus, EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.

Summary: Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease. : Shikatani et al. demonstrate that the nuclear transcription factor c-Myb exacerbates experimental atherosclerosis directly through its effects on B lymphocytes. Paradoxically, c-Myb promotes B2 cell development yet limits numbers of IgM-producing antibody-secreting cells and levels of atheroprotective OxLDL-specific IgM antibodies.

Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

The regulated response of endothelial cells to signals in their environment is not only critical for the de novo formation of primordial vascular networks during early development (ie, vasculogenesis), but is also required for the subsequent growth and remodeling of new blood vessels from preexisting ones (ie, angiogenesis). Vascular endothelial growth factors (Vegfs) and their endothelial cell-specific receptors play a crucial role in nearly all aspects of blood vessel growth. How the outputs from these pathways affect and coordinate endothelial behavior is an area of intense research. Recently, numerous studies have highlighted roles for microRNAs in modulating Vegf signaling output in several different contexts. In this review, we will provide an overview of how small RNAs regulate multiple aspects of the Vegf signaling pathway. In particular, we highlight areas where identification of microRNAs and their targets has provided new insight into the role of downstream effectors in modulating Vegf output during development. As Vegf plays a broad role in multiple aspects of endothelial biology and has become a target for therapeutic manipulation of pathological blood vessel growth, microRNAs that affect Vegf signaling output will undoubtedly be major targets of clinical value.

Atherosclerosis is a vascular pathology characterized by inflammation and plaque build-up within arterial vessel walls. Vessel occlusion, often occurring after plaque rupture, can result in myocardial and cerebral infarction. Epigenetic changes are increasingly being associated with atherosclerosis and are of interest from both therapeutic and biomarker perspectives. Emerging genomic approaches that profile DNA methylation, chromatin accessibility, post-translational histone modifications, transcription factor binding, and RNA expression in low or single cell populations are poised to enhance our spatiotemporal understanding of atherogenesis. Here, we review recent therapeutically relevant epigenetic discoveries and emerging technologies that may generate new opportunities for atherosclerosis research.